This application is a first resubmission of a Phase I application. An area of special interest to NIAID for SBIR/STTR applications is "Biomarkers of Host Immune Defense to Bioterrorism Pathogens". The use of microRNA's (miRNA) expressed in white blood cells as biomarkers to screen for infection is novel and potentially very powerful because not only do host cell miRNA's change upon infection, but both RNA and DNA viruses regulate miRNA that are expressed in the host cell. Even more novel is an assay that measures both miRNA and mRNA biomarkers simultaneously. This Phase I application will develop and validate a lysis-only, quantitative and highly reproducible high density array miRNA platform (the HD qNPA miRNA assay) based on adapting a nuclease protection assay of mRNA gene expression, the quantitative Nuclease Protection Assay (qNPA") marketed by HTG, to measure miRNA. Since the first submission, HTG has adapted the qNPA mRNA assay to a high density array platform and is in the process of validating a qNPA-base custom microplate miRNA assay. This assay will be adapted to the high density platform and validated in this Phase I program. qNPA only requires sample lysis (no extraction of the mRNA), making this a novel approach to miRNA quantification. Current methods for measuring miRNA are problematic, in part because of uncertainties regarding the efficiency of extraction, the difficulty current methods have measuring mRNA (much less miRNA) from fixed tissue, and the poor quantitative performance of current high density arrays and miRNA measurement methods. Performance of the HD qNPA miRNA assay will be first validated for samples of cells, fixed tissues, whole blood, bronchial lavage, sputum, and tracheal aspirates. The expectation is that whole assay CV's will be <20% (ideally <10%) from these complex clinical samples, enabling the identification of changes of <1.4-fold (ideally <1.2-fold), and that sensitivity will enable the use of <60 ml whole blood or as few as 5,000 cells. A training set of blood samples from patients diagnosed with sepsis and a training set of archived formalin fixed paraffin embedded biopsies from patients with diffuse large B cell lymphoma (DLBCL), who subsequently received chemotherapy plus anti-CD20 monoclonal Ritixan, will be tested. These two clinical studies will validate the miRNA discovery concept of the platform and demonstrate that quantitative differential profiles of miRNA can be identified from such samples (compared to normal or control samples), providing sets of putative miRNA biomarkers useful as a predictive screen for response to therapy (in the case of DLBCL) or for initiating "early goal-directed therapy" (in the case of sepsis). In Phase II, test sets of samples for DLBCL and sepsis will be evaluated, and the method will be extended to identify and validate white cell miRNA signatures useful for screening for infection by Category A, B, and C agents, addressing whether organism-specific signatures can be identified, or whether the miRNA signatures will be specific for the class of pathogen, or only generic for infection. The identified miRNA will also be evaluated for utility as targets for anti-infectives. PUBLIC HEALTH RELEVANCE: This 2-year Phase I STTR application involving HTG and the University of Arizona is being resubmitted (for the first time) to address an area of special interest to NIAID for "Biomarkers of Host Immune Defense to Bioterrorism Pathogens". We propose to develop and validate a lysis-only, quantitative and highly reproducible high density array microRNA (miRNA) platform - the HD qNPA miRNA assay - based on adapting a nuclease protection assay of mRNA gene expression, the microplate-based quantitative Nuclease Protection Assay (qNPA") marketed by HTG. We propose to demonstrate that this novel HD qNPA miRNA platform can identify sets of miRNA from host cells within clinical samples (blood, bronchial lavage, sputum, tracheal aspirate, formalin fixed paraffin embedded tissue), and to specifically validate performance as a screening method for sepsis and diffuse large B cell lymphoma. The use of miRNA as biomarkers of infectious diseases is novel and potentially very powerful since not only do host cell miRNA's change upon infection, but both RNA and DNA viruses regulate miRNA that are expressed in the host cell. Additionally, other significant and novel aspects of this application are that the assay will measure both miRNA and mRNA signatures, that it will not require extraction (problematic for miRNA), and that it should provide superior performance in terms of sensitivity, precision, reproducibility and ability to measure miRNA from clinically relevant samples (such as whole blood and fixed tissue). In Phase II, this platform will be applied to identify host cell miRNA signatures useful for screening for infection by Category A, B and C pathogens, and useful as targets for drug discovery.